CN117327726A - Application of OpCBR1 transcription factor in improving hairy root growth and camptothecin production of Snakeroot grass - Google Patents

Abstract

The invention provides the application of an OpCBR1 transcription factor in improving the growth of the hairy roots of Snakeroot and increasing the production of camptothecin. By constructing a plant expression vector containing the OpCBR1 gene and genetically transforming the explant of the Snakeroot stem segment, it is obtained Hairy roots of short snakeroot overexpressing the OpCBR1 gene; the biomass and camptothecin production of the transgenic hairy roots were analyzed by weighing method and high performance liquid chromatography. The results showed that overexpression of the OpCBR1 gene could improve short snakeroot by increasing The biomass of the hairy roots of grass thus increased its camptothecin production; through dynamic observation, it was found that the biomass of the hairy roots of the OpCBR1 transgenic line increased significantly on the 16th day and reached the maximum on the 32nd day. At the same time, the yield also reached the maximum value, indicating that overexpression of the OpCBR1 gene can significantly shorten the culture period. The invention provides a new high-quality drug source for producing camptothecin with broad-spectrum anti-cancer efficacy, and has important theoretical significance and potential application value.

Description

OpCBR1 transcription factor improves hairy root growth and increases camptothecin in Snakeroot Applications in production

Technical field

The invention belongs to the field of genetic engineering technology and relates to the application of an OpCBR1 transcription factor in increasing the growth of hairy roots of Snakeroot and increasing the production of camptothecin.

Background technique

Ophiorrhiza pumila belongs to the genus Ophiorrhiza of the Rubiaceae family. It is a perennial dicotyledonous herb and one of the medicinal plants that produce camptothecin. Camptothecin is an important natural product with broad-spectrum anti-cancer activity, and its derivatives are often used clinically to treat colon cancer, rectal cancer, ovarian cancer, etc. However, a single planting model and scarce germplasm resources have severely restricted the supply of camptothecin anticancer drugs and cannot meet the growing market demand year by year. Therefore, improving the production of camptothecin has become a research focus in recent years. It is an effective method to improve the production of secondary metabolites in plants through metabolic engineering technology. In recent years, transcription factors have become a research hotspot and have been widely reported to be involved in regulating plant secondary metabolism.

The ERF family is one of the largest families of transcription factors in plants. Its unique DNA-binding domain consists of three β-strands and α-helices. This domain consists of about 60 to 70 amino acids and is involved in DNA binding and can recognize specific Cis-regulatory elements, including GCC-box elements (5'-AGCCGCC-3'). The ERF family is further divided into two main subfamilies, including the ERF subfamily and the DREB subfamily. ERF family transcription factors play an important regulatory role in many biological and physiological processes such as plant morphogenesis, various stress response mechanisms, hormone signal transduction, and metabolite regulation.

Based on the powerful anti-tumor activity of camptothecin and the growing market demand year by year. In order to ensure an adequate supply chain of camptothecin, there is an urgent need to find alternative drug sources and increase the production of camptothecin in drug sources. At the same time, Snakeroot grass has the characteristics of short growth cycle, easy genetic transformation, and has a mature tissue culture system. It is a good source of in vitro production of camptothecin. In addition, the ERF family plays a role in participating in plant growth and regulating metabolites.

Contents of the invention

The present invention provides the coding sequence of the OpCBR1 transcription factor of Snakeroot and its application. The OpCBR1 of Snakeroot can promote the growth of the hairy roots of Snakeroot and increase the production of camptothecin.

The present invention can be realized through the following technical solutions:

The present invention clones the OpCBR1 transcription factor from Snakeroot. The nucleotide sequence of the OpCBR1 transcription factor is as shown in SEQ ID NO. 1, and its gene coding frame sequence is 582 bp; the amino acid sequence of the OpCBR1 transcription factor is as follows Shown in SEQ ID NO.2. An overexpression vector was constructed, and transgenic hairy roots were obtained by genetic transformation of Snakeroot stem segments; the production of camptothecin in the transgenic hairy roots was determined by high-performance liquid chromatography.

The present invention also provides the application of the above-mentioned Snakeroot OpCBR1 transcription factor in improving the hairy root growth and camptothecin production of Snakeroot, and overexpressing the OpCBR1 transcription factor in Snakeroot.

Further, the overexpression of the OpCBR1 transcription factor in Heteroporum brevis is specifically:

Transform Agrobacterium rhizogenes with a plant overexpression vector containing the OpCBR1 transcription factor to obtain an Agrobacterium rhizogenes strain having the plant overexpression vector;

The constructed Agrobacterium rhizogenes strain is used to transform the explants of Snakeroot. Resistant hairy roots are obtained through antibiotic screening. The hairy root strains that are positive after PCR detection are overexpressed in Snakeroot. Transgenic snakeroot hairy roots for the OpCBR1 transcription factor.

Further, the plant overexpression vector containing OpCBR1 transcription factor includes the nucleotide sequence shown in SEQ ID NO.1. Plant overexpression vectors can be prepared by conventional methods in the art, such as by constructing the nucleotide sequence of the OpCBR1 transcription factor into an expression vector, which includes plasmids and the like.

The invention also provides a method for using the OpCBR1 transcription factor to increase the growth of the hairy roots of Snakeroot and increase the production of camptothecin, which includes the following steps:

Step 1. Operably connect the OpCBR1 transcription factor to the expression control sequence to form a plant overexpression vector containing the OpCBR1 gene; wherein the nucleotide sequence of the OpCBR1 transcription factor is shown in SEQ ID NO.1;

Step 2. Transform Agrobacterium rhizogenes with the plant overexpression vector containing the OpCBR1 gene to obtain the Agrobacterium rhizogenes strain having the plant overexpression vector;

Step 3. Use the Agrobacterium rhizogenes strain constructed in step 2 to transform the explants of Snakeroot grass, and obtain resistant hairy roots through antibiotic screening. The hairy root lines that are positive by PCR are considered to be transgenic short snakeroots. Snakeroot hairy roots, transgenic short snakeroot hairy roots have faster growth rate and higher camptothecin production.

In step 2, the freeze-thaw method is used for transfer, and the Agrobacterium rhizogenes is C58C1 strain.

In one embodiment of the present invention, in step 2, the OpCBR1 gene is constructed into pHB-X-YFP to obtain a plant overexpression vector containing the OpCBR1 gene, wherein the plant expression vector used is the modified pHB-X-YFP vector, Contains p35S promoter and HYG terminator, multiple cloning site, origin of replication and kanamycin resistance site.

In one embodiment of the present invention, the PCR detection method described in step 3 is as follows: design specific primers for the hairy root gene rolB and perform PCR amplification; design specific primers for the CaMV35S promoter and the upstream and downstream inserted gene OpCBR1 and perform PCR amplification ; Agarose gel electrophoresis detection, if the target band appears, it is a positive clone.

In one embodiment of the present invention, in step 2, the resistant hairy roots obtained through antibiotic screening are specifically:

Place the transformed snakeroot explants on the surface of B5 culture medium, and cultivate them in the dark at 28°C for 2 days. Then transfer them to the first-level sterilized solid medium, cultivate them in the dark at 25°C, and replace them every 2 weeks. Primary culture medium until hairy roots grow from the callus of the explant, then transfer to the secondary sterilized solid medium for dark culture at 25°C until the hairy roots grow to 3-4cm and begin to branch. At this time, the hairy root clones with good growth and branches were cut from the callus and transferred to the third-level sterilization solid medium for dark cultivation at 25°C; after culturing for a period of time, they were cultured in the third-level sterilization solid medium. Select clones with good growth status and no overflow phenomenon on bacterial solid medium. Cut the newly grown hairy roots with a tip of about 2cm from these clones and transfer them to B5 solid medium. Continue dark culture at 25°C to obtain resistant hairy roots; the first-level sterilization solid medium is B5+Cb 300mg/L, the second-level sterilization solid medium is B5+Cb 200mg/L, and the third-level sterilization solid culture is The base is B5+Cb 100mg/L.

In one embodiment of the present invention, high-performance liquid chromatography is used to determine the camptothecin content in the hairy roots of transgenic Snakeroot grass. The high-performance liquid chromatography method is as follows: 20 μL of camptothecin is taken from each transgenic strain. The crude extract was injected into a high-performance liquid chromatograph. The chromatographic conditions used are: the chromatographic column uses a C-18 reversed-phase silica gel column, with the ratio of acetonitrile: water = 35:65 (V: V) as the mobile phase, the column temperature is set to 30°C, and the flow rate is 1mL/min. The detection wavelength is 254nm.

The invention discloses a metabolic engineering method for OpCBR1 transcription factor to increase the growth of hairy roots of Snakeroot and increase the production of camptothecin. The present invention clones an OpCBR1 transcription factor from the medicinal plant Snakeroot, and constructs its subcellular localization vector; by constructing plant overexpression and suppression expression vectors, genetically transforms Snakeroot stem segment explants, and obtains transgenic hairy roots. ; The biomass was detected through observation on different days; the biomass of the hairy roots of the OpCBR1 transgenic line increased significantly on the 16th day, reaching the maximum value on the 32nd day, and the yield also reached the maximum value. The HPLC result analysis was satisfactory. The strain with the highest dendritic acid production was 0.51 mg/bottle, which was 2.04 times that of the control group. The results showed that overexpression of the OpCBR1 gene could significantly shorten the culture period, and overexpression of the OpCBR1 transcription factor would increase the biomass of hairy roots and increase camptothecin production. It provides a new high-quality drug source for the production of camptothecin with broad-spectrum anti-cancer efficacy, which has important theoretical significance and potential application value.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

Figure 1 shows the expression level of OpCBR1 detected by fluorescence real-time quantitative PCR in the hairy roots of Snakeroot overexpressing the OpCBR1 gene in the embodiment of the present invention, showing that the expression level of the OpCBR1 gene is increased;

Figure 2 shows the dry weight DW and fresh weight FW levels of the hairy roots of Snakeroot overexpressing the OpCBR1 gene measured on the 32nd day in the example of the present invention, showing that the biomass is significantly higher than that of the control group;

Figure 3 is an example of the present invention using HPLC to detect the production of camptothecin CPT in the hairy roots of Snakeroot overexpressing the OpCBR1 gene on the 32nd day, showing that the production of camptothecin is increased;

Figure 4 is a dynamic measurement of the dry weight level of the hairy roots of Snakeroot overexpressing the OpCBR1 gene in the embodiment of the present invention, showing that the biomass increases with time;

Figure 5 is an example of the present invention using HPLC to dynamically detect the production of camptothecin in the hairy roots of Snakeroot overexpressing the OpCBR1 gene, showing that overexpressing the OpCBR1 gene can significantly shorten the cultivation cycle.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims of this application. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in molecular cloning (Sambrook et al.), or the conditions recommended by the instructions provided by the manufacturer or the instructions accompanying the kit.

Example 1: Cloning of Snakeroot OpCBR1 gene

1. Extraction of total RNA from Snakeroot

Take a small amount of young snakeroot leaves, quickly freeze them in liquid nitrogen, quickly grind them in a mortar, add them to a 1.5mL Eppendorf (EP) centrifuge tube containing the lysis solution, shake them thoroughly, and extract them according to the instructions of the TIANGEN kit. Extract total RNA. Check RNA quality using agarose gel electrophoresis, and then measure RNA content on a spectrophotometer.

2. Cloning of the OpCBR1 gene of Snakeroot

Using the extracted total RNA as a template, cDNA was synthesized after reverse transcription; gene-specific primers were designed according to the sequence of the OpCBR1 gene, as shown in Table 1. The OpCBR1 gene was amplified from the total cDNA through PCR amplification and sequenced.

Table 1 PCR primers

Primer name Primer sequence (5'-3') OpERF20-FP(SEQ ID NO.3) ATGATCTCAATCTCTGATCT OpERF20-RP(SEQ ID NO.4) TCAAGTACTTTTCCTTCTTTT

Through the above steps, the full-length coding sequence (SEQ ID NO. 1) of the transcription factor in Heliconia brevis was obtained and its protein coding sequence (SEQ ID NO. 2) was deduced, in which the start codon is ATG, The stop codon is TGA.

Example 2: Construction of plant overexpression vector containing OpCBR1 gene

The OpCBR1 gene was constructed on the plant expression vector pHB-X-YFP. In order to facilitate the construction of the expression vector, the BamHI cleavage site was introduced into the forward primer, and the Spe I cleavage site was introduced into the reverse primer. Primers are shown in Table 2;

Table 2 PCR primers for pHB-OpCBR1-YFP vector construction

Primer name Primer sequence (5'-3') OpCBR1-BamHI-FP(SEQ ID NO.5) CCAGTCTCTCTCCAAGCTTGGATCCATGATCTCAATCTC OpCBR1-SpeI-RP(SEQ ID NO.6) CGCCCTTGCTCACCATACTAGTAGTACTTTTCCTCT

In this implementation example, the transcription factor OpCBR1, which is involved in regulating the synthesis of camptothecin, is operably connected to the expression control sequence, and a plant overexpression vector pHB-OpCBR1-YFP containing the OpCBR1 gene is constructed. This expression vector can be used to achieve metabolic engineering through metabolic engineering strategies. Increase the camptothecin content in snakeroot.

Example 3: Agrobacterium rhizogenes-mediated OpCBR1 overexpression vector genetically transforms Snakeroot grass to obtain transgenic hairy roots

1. Obtaining engineered Agrobacterium rhizogenes containing overexpression of OpCBR1 gene

The plant expression vector containing the OpCBR1 gene in Example 2 was transferred into Agrobacterium rhizogenes (such as C58C1, which is a biological material publicly sold in the market) using the freeze-thaw method, and PCR verification was performed.

2. Agrobacterium rhizogenes mediates the genetic transformation of OpCBR1 gene into Snakeroot grass

2.1 Pre-culture of explants

Cut stem segments from strong sterile snakeroot seedlings, place them on B5 medium for pre-culture, and culture them in the dark at 25°C for 2 days.

2.2 Co-culture of Agrobacterium and explants

Transfer the explants of the short snakeroot stem segments into the B5 culture medium suspension of Agrobacterium rhizogenes containing the activated OpCBR1 gene overexpression vector (OD value is about 0.3) and soak for 10 minutes, Shake gently to fully contact the explant with the bacterial liquid. After the infection is completed, take out the short snakeroot stem segment and place it on the surface of sterile absorbent paper, absorb the bacterial liquid, and then place it on the B5 culture medium. On the surface, incubate in the dark at 28°C for 2 days.

2.3 Induction and subculture of hairy roots

After the above materials were cultured in the dark for 2 days, they were transferred to a first-level sterilized solid medium (B5+Cb300mg/L) and cultured in the dark at 25°C. The culture medium was replaced approximately every 2 weeks. After about 14-20 days, small callus will appear in the wound of the above-mentioned explant. After that, light yellow hairy roots will gradually grow in the callus. At this time, transfer the stem segments that have grown hairy roots, and transfer them in batches to the secondary sterilized solid medium (B5+Cb200mg/L). On this medium, the hairy roots can grow faster. growth. After 2-3 weeks, the hairy roots grow to 3-4cm and begin to branch. At this time, the hairy root clones with good growth and branches are cut from the callus and transferred to three On grade sterilized solid medium (B5+Cb 100mg/L), culture was still carried out in the dark at 25°C. After culturing for a period of time, select clones that are in good growth condition and have no overflow phenomenon on the third-level sterile solid medium. Cut the newly grown hairy roots with a tip of about 2cm from these clones. , transfer it to B5 solid medium and continue culturing. The culture conditions are dark culture at 25°C. After about 2 weeks of culture, strains without overflow were selected for independent numbering.

3. PCR detection of transgenic snakeroot hairy roots

As shown in Table 3, according to the 35S promoter region and OpERF20 upstream of the expression cassette where the target gene is located, the forward primer (SEQ ID NO.9) and the reverse primer (SEQ ID NO.10) were respectively designed to perform PCR detection of the target gene; At the same time, the upstream and downstream primers (SEQ ID NO. 7-8) of the RolB gene expressed in hairy roots were used to conduct PCR detection of the RolB gene. A total of 30 monoclonal hairy root lines were identified, of which 12 were positive lines.

Table 3 PCR primers for identification of OpCBR1 transgenic hairy root lines

Primer name Primer sequence (5'-3') RolB-F(SEQ ID NO.7) GCTCTTGCAGTGCTAGATTT RolB-R(SEQ ID NO.8) GAAGGTGCAAGCTACCTCTC OpCBR1-F243(SEQ ID NO.9) GGGGACTGGACAAGGTATCG PHB-R(SEQ ID NO.10) TGTGGCCGTTTACGTCGC

In this example, the plant expression vector is transformed into Agrobacterium rhizogenes to obtain the Agrobacterium rhizogenes strain containing the OpCBR1 gene plant overexpression vector used to transform Snakeroot grass, and the Agrobacterium rhizogenes is used to infect Snakeroot root. Grass stem segments were used to obtain transgenic short snakeroot hairy roots tested by PCR. The acquisition of transgenic Snakeroot hairy roots provides direct material for screening the hairy roots of Snakeroot with higher camptothecin content.

Example 4: Determination of Camptothecin Content in Transgenic Snakeroot Hairy Root Lines Using HPLC

1. Hairy root liquid culture

Select the hairy roots with good growth status in Example 3, cut the middle and tip, and inoculate it into 100 mL of B5 liquid culture medium. Then use a shaker to expand the culture, and culture it in the dark at 25°C and 100 rpm. . The hairy roots are harvested after about 45 days of cultivation. Since camptothecin is excreted, two parts, the culture medium and the hairy roots, need to be harvested. Part 1: Harvest of the culture medium. After taking out the hairy roots from the Erlenmeyer flask, first measure the volume of the culture medium, then rotary evaporate the culture medium, and finally dilute the volume to 2 mL, and store it at 4°C for subsequent use of Camptotheca acuminata. Determination of alkali content; Part 2: Harvest of hairy roots, take out the hairy roots from the culture container, and then use absorbent paper to dry the surface culture medium. First, cut 3 portions of hairy roots, each weighing approximately 0.2 -0.6g, wrapped and labeled in tin foil, quickly frozen in liquid nitrogen, and then stored at -80°C for subsequent RNA extraction. The remaining hairy roots were wrapped and labeled with absorbent paper, and then placed in a 50°C constant temperature oven to dry. When there was no change in weight, they were taken out and used for subsequent extraction and content determination of camptothecin.

2. Extraction of camptothecin from hairy roots

Take out the hairy roots that have been dried to constant weight, grind them into powder, weigh 0.1g of the powder, and put it into a 50mL centrifuge tube; add a volume of about 20mL of chromatographically pure methanol, cover the centrifuge tube tightly, and seal it with a sealing film Keep, vortex for 3 minutes to mix, and then place it in an ultrasonic water bath for extraction for 1 hour. Then place the ultrasonic mixture in a 50°C oven and let it stand overnight; centrifuge it in a centrifuge at 12,000 rpm for 10 minutes the next day. , take the supernatant after centrifugation, evaporate the supernatant to dryness with a vacuum rotary evaporator, set the temperature to 70°C during evaporation, redissolve it with chromatographically pure methanol after evaporation, and finally adjust the volume to 2 mL, and then filter it. Filter through a 0.22 μm organic phase filter. After filtration, the camptothecin crude extract is obtained and stored at -20°C for later use.

3. qRT-PCR detection of transgenic snakeroot hairy roots

RNA was extracted from the hairy roots of transgenic Snakeroot and reverse transcribed into cDNA. The OpCBR1 gene was analyzed using qRT-PCR. The results showed that the expression of OpCBR1 gene was increased, and OpCBR1-OE-26 and OpCBR1-OE-27 were increased by fold. highest (see Figure 1), these two strains were selected for subsequent biomass determination.

4. Determination of fresh weight and dry weight of transgenic hairy root lines

The transgenic hairy roots were harvested at different times and were photographed to record the hairy root phenotype. The fresh weight of the hairy roots was measured after absorbing water from the hairy roots with absorbent paper. The dry weight was measured after freeze-drying in a freeze dryer. Results: The dry weight and fresh weight of OE-26 and OE-27 strains during the entire dynamic measurement period were higher than those of the unloaded control strain pHB-EV (see Figures 2 and 4), indicating that OpERF1 overexpression strains will affect Growth of hairy roots, increasing biomass.

5. HPLC determination of camptothecin content in hairy roots

First, configure the camptothecin standard and create a standard curve. The preparation method of camptothecin standard is as follows: Use an analytical balance to weigh 1 mg of camptothecin standard, place it in a small beaker, add 10 mL of methanol to the beaker, and then ultrasonicate the small beaker in the water bath of the ultrasonic device for 10-20 minutes. After dissolution, it is the standard solution with a final concentration of 100 μg/mL. When measuring the standard, dilute it into multiple different concentration gradients to create a standard curve.

Take 20 μL of each camptothecin crude extract and inject it into a high-performance liquid chromatograph. The chromatographic column used was a C-18 reversed-phase silica gel column (Symmetry Shield TM C18, 5 μm, 250x4.6 mm, Waters); the chromatographic conditions were: mobile phase acetonitrile: water = 35:65; column temperature 30°C; flow rate set to 1mL/min; the detection wavelength is set to 220nm. After the detection, record the peak area of the camptothecin component of each sample in HPLC, substitute the peak area into the above-mentioned standard curve, and calculate the camptothecin content in the sample. The results show that based on the measured content and dry weight and fresh weight levels at different days, it was found that the yield of the overexpressed transgenic line OpCBR1 can reach the level of 36D at 32D, which can significantly shorten the cultivation period (see Figure 3 and Figure 5). In the present invention, compared with the control, the camptothecin production and biomass in the transgenic hairy root line overexpressing OpCBR1 were significantly increased. Among them, the line with the highest camptothecin production was 0.51 mg/bottle, which was 2.04 of the control group. times.

The preferred embodiments of the present invention are described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, any technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention and on the basis of the prior art should be within the scope of protection determined by the claims.

Claims (8)

1. The application of the OpCBR1 transcription factor in improving the growth of hairy roots of the short-snake root grass and increasing the output of camptothecine is characterized in that the OpCBR1 transcription factor is overexpressed in the short-snake root grass, and the amino acid sequence of the OpCBR1 transcription factor is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the overexpression of OpCBR1 transcription factor in belleville grass is specifically:

transforming agrobacterium rhizogenes with a plant overexpression vector containing the OpCBR1 transcription factor to obtain agrobacterium rhizogenes strains with the plant overexpression vector;

transforming the hairy root grass explant by using the constructed agrobacterium rhizogenes strain, screening by antibiotics to obtain a resistant hairy root, and detecting the hairy root strain positive by PCR to obtain the transgenic hairy root of the short snake root, wherein the transgenic short snake root is used for over-expressing the OpCBR1 transcription factor in the short snake root.

3. The use according to claim 2, wherein the plant overexpression vector containing the OpCBR1 transcription factor comprises the nucleotide sequence as set forth in SEQ ID No. 1.

4. The use according to claim 2, wherein the agrobacterium rhizogenes is a C58C1 strain.

5. The use according to claim 2, wherein the antibiotic screening to obtain resistant hairy roots is specifically:

placing the transformed short-snake root grass explant on the surface of a B5 culture medium, carrying out dark culture at 28 ℃ for 2 days, transferring to a first-stage degerming solid culture medium, carrying out dark culture at 25 ℃ and replacing the culture medium every 2 weeks until hairy roots grow out on the callus of the explant, transferring to a second-stage degerming solid culture medium, carrying out dark culture at 25 ℃ until the hairy roots grow to 3-4cm, and beginning to generate branches, cutting off the hairy root monoclone with better growth vigor and branches from the callus, and transferring to a third-stage degerming solid culture medium for dark culture at 25 ℃; after culturing for a period of time, selecting clone lines which have good growth state and do not have bacteria overflowing phenomenon on a three-stage sterilization solid culture medium, cutting off newly-grown tip hairy roots from the clone lines, transferring the hairy roots into a B5 solid culture medium, and continuing to perform dark culture at 25 ℃ to obtain resistant hairy roots; wherein the primary sterilization solid culture medium is B5+Cb300mg/L, the secondary sterilization solid culture medium is B5+Cb200 mg/L, and the tertiary sterilization solid culture medium is B5+Cb100 mg/L.

6. A method for increasing growth of hairy roots of pumice roots and increasing camptothecin production using an OpCBR1 transcription factor, comprising the steps of:

step 1, operatively connecting an OpCBR1 transcription factor with an expression regulatory sequence to form a plant over-expression vector containing an OpCBR1 gene; wherein the nucleotide sequence of the OpCBR1 transcription factor is shown in SEQ ID NO. 1;

step 2, transforming agrobacterium rhizogenes with a plant over-expression vector containing an OpCBR1 gene to obtain agrobacterium rhizogenes strains with the plant over-expression vector;

step 3, transforming the short-snake root grass explant by utilizing the agrobacterium rhizogenes strain constructed in the step 2, obtaining a resistant hairy root through antibiotic screening, and the hairy root strain which is positive through PCR detection is the hairy root of the transgenic short snake root grass, and the hairy root of the transgenic short snake root grass has higher growth speed and higher camptothecin yield.

7. The method according to claim 6, wherein in the step 2, the agrobacterium rhizogenes is C58C1 strain, and the transfer is performed by a freeze thawing method.

8. The method according to claim 6, wherein in the step 3, the step of obtaining the resistant hairy root by antibiotic screening is specifically:

placing the transformed short-snake root grass explant on the surface of a B5 culture medium, carrying out dark culture at 28 ℃ for 2 days, transferring to a first-stage degerming solid culture medium, carrying out dark culture at 25 ℃ and replacing the culture medium every 2 weeks until hairy roots grow out on the callus of the explant, transferring to a second-stage degerming solid culture medium, carrying out dark culture at 25 ℃ until the hairy roots grow to 3-4cm, and beginning to generate branches, cutting off the hairy root monoclone with better growth vigor and branches from the callus, and transferring to a third-stage degerming solid culture medium for dark culture at 25 ℃; after culturing for a period of time, selecting clone lines which have good growth state and do not have bacteria overflowing phenomenon on a three-stage sterilization solid culture medium, cutting off newly-grown tip hairy roots from the clone lines, transferring the hairy roots into a B5 solid culture medium, and continuing to perform dark culture at 25 ℃ to obtain resistant hairy roots; wherein the primary sterilization solid culture medium is B5+Cb300mg/L, the secondary sterilization solid culture medium is B5+Cb200 mg/L, and the tertiary sterilization solid culture medium is B5+Cb100 mg/L.